It is recognized that particles are available to which biological molecules may be attached. U.S. Pat. No. 4,438,239 describes microbeads between 100 and 2000 angstroms in diameter containing active aldehydes which form conjugates with proteins and enzymes. U.S. Pat. No. 4,035,316 describes microbeads less than 3 microns in diameter to which lectins or antibodies may be bound for use in cell separation. U.S. Pat. Nos. 4,157,323 and 4,170,685 describe impregnated functional microbeads which can be covalently bound to antibodies and used to separate cells. The described microbeads are not suitable for simulating biological cells since they are smaller than biological cells which range generally from 7-15 microns in diameter. Moreover, the described microbeads are not highly uniform and have coefficients of variation greater than five percent (5%) in diameter. More specifically, so far as applicant is aware no one has heretofore realized the possibility of covalently binding functional biological molecules to uniform microbeads for biological cell simulation.
Recently, larger highly uniform microbeads with coefficients of variation of one percent (1%) in diameter have been synthesized both in the laboratory and in outer space. Such microbeads are described in NASA publication TM78132 "Large-size Monodesperse Latexes as Commercial Space Product", in U.S. Pat. Nos. 4,247,434 and in 4,336,173. Improvements to the method described in U.S. Pat. No. 4,336,173 are described in applicant's copending applications Ser. No. 685,464 entitled "Calibration Method for Flow Cytometry using Fluorescent Microbeads and Synthesis Thereof" and Ser. No. 805,654 entitled "Fluorescent Calibration Microbeads Simulating Stained Cells". None of these larger, uniform microbeads have been described or recognized as being useful for covalently binding antibodies or enzymes in cell simulation. However, the present invention recognizes that the microbeads described in applicant's copending applications, although used as fluorescent standards for flow cytometry and fluorescence microscopy, are capable of binding proteins for biological cell simulation because of functional groups introduced on the surface. Several examples are described in applicant's copending applications of protein binding by allowing the epoxy groups on the microbead surface to react with the primary amines of protein such as avidine and those associated with phycoerythrins.
In one aspect, the simulated cells of the invention address a method of screening the specificity of fluorescent antibodies and determining the number of fluorescent dye molecules which are conjugated to antibody molecules. In another aspect, the simulated cells of the invention are recognized as having potential uses in enzyme kinetics and chelation studies. Background with respect to these aspects of the invention is next described.
One known method for screening the specificity of antibodies has been to use diffusion-precipitation techniques where different concentrations of the antigen and antibody are diffused towards each other in gells and the degree of specificity being determined by finding the lowest concentration that allows the antigen and antibody to form a visible precipitate. Another method for screening antibodies is to use an inhibition test as in hemoagglutination and ELISA (Enzyme-Linked Immunosorbent Assay) testing. Here the antibody to be screened, e.g., a mouse IgM monoclonal, is reacted with a specific antigen immobilized onto a substrate, e.g. rabbit anti mouse IgM antibody on nitro cellulose paper. Then a fluorescenated or radioactive antibody, e.g., goat anti rabbit antibody, is exposed to the surface binding the mouse IgM antibody. If no goat anti rabbit antibodies bind to the surface as determined by fluorescence or radioactivity, then all binding sites are known to be specifically tied up by the mouse IgM antibody, proving that the mouse IgM antibody is very specific. Although this methodology is common, there is no known suitable surface which can be used in conjunction with a flow cytometer for such determination with respect to size and uniformity. More specifically, the art has not provided a substrate in suspension and of the type provided by the present invention exhibiting surface characteristics and a size comparable to biological cells suited to flow cytometry techniques.
One of the important aspects of immunology and cell biology is determining the number and density of antibody binding sites on a cell. At present this determination is both difficult and subject to gross errors without careful considerations of correction factors. For example, in using fluorescence to determine this number, the key is to first determine the number of fluorescent molecules, e.g., Fluorescein Isothiocyanate, designated as FITC, which have been conjugated to an antibody molecule. This determination is commonly carried out by measuring the absorption of a solution of the antibody at 365 nm to determine the antibody concentration and then again at 490 nm (with some correction factors for the absorption at 365 nm) to determine the fluorescent dye molecule concentration. By dividing the number of fluorescent dye molecules by the number of antibody molecules, one determines the number of dye molecules per antibody molecule, commonly referred to as the F/P ratio. However, as pointed out in the technical brochure "Quantitative Fluorescein Microbead Standards", published by Flow Cytometry Standards Corporation, this F/P ratio determined by absorption may be useless when working with fluorescence because it is well recognized that when dye molecules, e.g., FITC, are close together, their fluorescence may be quenched by each other resulting in less of a fluorescence signal than anticipated. Therefore, it is necessary to measure the effective fluorescence of the dye on the antibodies in terms of equivalent soluble dye molecules. The referred-to technical brochure describes how to do this by first measuring the fluorescence in a fluorometer, e.g., for FITC; excitation at 488 nm, emission at 520 nm and then the concentration of the antibody by absorption at 365 nm as before. The problem with this method is that it requires a fluorometer and calibration plots for the absorption and the fluorescence measurements.